The formation of lipid-laden macrophage foam cells within the arterial intima is a hallmark of atherosclerosis. The foamy appearance of these cells is due to the accumulation of cytoplasmic lipid inclusions containing cholesterol esters (CE). Loss of homeostatic balance between cholesterol uptake and efflux pathways is central to foam cell formation. While uptake of native or modified lipoproteins constitute the influx pathways, transfer of unesterified cholesterol from these foam cells to acceptors like high-density lipoproteins (HDL) is the only recognized mechanism for cholesterol efflux. The obligatory first step for the removal of cholesterol from foam cells via HDL is the hydrolysis of stored CE to release free or unesterified cholesterol (UC). UC then moves to the plasma membrane for transfer to the acceptor molecule. Neutral Cholesterol ester hydrolase (CEH) catalyzes the hydrolysis of stored cholesterol esters. The long-term objective of this research is to determine the mechanisms involved in the regulation of CEH in human macrophage and how these regulatory processes can be manipulated to affect macrophage foam cell formation and/or regression. It is the central hypothesis of this proposal that: Macrophage CEH levels affect HDL-mediated cholesterol efflux from foam cells and that expression of CEH is modulated by processes affecting the levels of cellular cholesterol and its metabolites. The following four Specific Aims are proposed: Aim 1: To determine changes in intracellular cholesterol ester metabolism by over-expression of human macrophage CEH cDNA: Effect on lipid droplet mobilization and cholesterol enrichment of plasma membrane. Aim 2: To determine changes in cholesterol efflux and resulting changes in cellular cholesterol/cholesterol esters by over-expression of human macrophage CEH cDNA and to examine the regulation of CEH by HDL-mediated signaling. Aim 3: To determine the mechanisms whereby intracellular cholesterol/oxysterols levels regulate CEH expression. Aim 4: To obtain in vivo "proof of concept" by introducing human macrophage CEH transgene in LDL-receptor knockout (LDLR-/-) mice and attenuating cholesterol-feeding induced atherosclerosis. While, the production of the desired phenotype in the transgenic mice may provide basis for future gene therapy, delineation of the regulatory mechanisms involved would identify potential targets for therapeutic intervention. Given the prevalence of atherosclerosis and coronary artery disease, the current findings are likely to have important clinical relevance.